It is recommended that infants are breast fed exclusively for the first six months of life; however, less than 15% of infants in the United States are meeting this recommendation and only 44% of infants receive human milk to any extent at six months of age (Gartner et al., 2005; Li et al., 2005; Eidelman et al., 2012). Nearly 40% of infants receive formula as their sole source of nutrition by one month of age (Li et al., 2005). Probiotics and/or prebiotics are increasingly supplemented in infant formulae and are generally regarded as safe in healthy infants without concern for growth or adverse effects (Holscher et al., 2012a; Holscher et al., 2012b). Additionally, human milk oligosaccharides have been shown to enhance intestinal maturation in vitro. Thus, we aimed to assess 1) the effects of the probiotic Bifidobacterium animalis subspecies lactis, 2) the prebiotics galacto- and fructo-oligosaccharides, and 3) the individual and combined effects of specific human milk oligosaccharides as means to narrow the compositional gap between human milk and infant formula. The central hypothesis of this research was that specific ingredients would enhance gastrointestinal health and development.
Addition of probiotics to infant formula may positively affect immune function in non-exclusively breastfed infants. Probiotics are live microorganisms that, when administered orally in adequate amounts, survive the digestion process and confer a benefit to the host (FAO/WHO, 2001). Our first study investigated the effects of an infant formula containing the probiotic Bifidobacterium animalis subspecies lactis (B. lactis) on intestinal immunity and inflammation (Holscher et al., 2012a). Six-week-old healthy, full-term infants (n=172) were enrolled in a prospective, randomized, double-blind, controlled clinical trial with 2 groups studied in parallel to a breastfed comparison group. Formula-fed (FF) infants were randomized to partially hydrolyzed whey formula (CON), or the same formula containing 106 CFU B.lactis/g (PRO) for 6 weeks. Fecal secretory IgA (sIgA), calprotectin, lactate, and stool pH were assessed at baseline, 2, and 6 weeks. Anti-poliovirus-specific IgA and anti-rotavirus-specific IgA in stool were assessed at 2 and 6 weeks. Among vaginally-delivered FF infants (n=39), PRO consumption increased (p<0.05) fecal sIgA, compared to CON. Anti-poliovirus-specific IgA concentration increased (p<0.05) in all infants consuming PRO; whereas anti-rotavirus-specific IgA tended to increase (p=0.056) with PRO consumption in cesarean-delivered infants (n=14). Anthropometrics and tolerance did not differ significantly between FF infants. In conclusion, infants consuming formula with B. lactis produced feces with detectable presence of B. lactis and augmented sIgA concentration. Furthermore, cesarean-delivered infants consuming B. lactis had heightened immune response, as evidenced by increased anti-rotavirus- and anti-poliovirus-specific IgA following immunization. These results demonstrate that negative immune-related effects of not breast feeding and cesarean delivery can be mitigated by including B. lactis in infant formula and thereby provide infants a safe, dietary, immune-modulating bacterial introduction.
As infant formula containing prebiotics may beneficially impact gastrointestinal tolerance and composition of the commensal microbiota, we assessed gastrointestinal tolerance and fecal microbiota, pH, and short-chain fatty acid (SCFA) concentrations of infants consuming formula with or without prebiotics in a second trial utilizing the a similar model as that discussed above (Holscher et al., 2012b). Prebiotics are selectively fermented ingredients that result in specific changes, in the composition and/or activity of the GI microbiota, thus conferring benefit(s) to the host (Gibson et al., 2010). FF were randomized to consume a partially hydrolyzed whey formula with (PRE) or without (CON) 4g/L of galacto-oligosaccharides and fructo-oligosaccharides (9:1) for 6 weeks. Fecal bacteria, pH, and SCFA were assessed at baseline, 3, and 6 weeks. Caregivers of subjects recorded stool characteristics and behavior for 2 days before the 3- and 6-week visits. Feces from infants fed PRE had a higher absolute number (p=0.0083) and proportion (p=0.0219) of bifidobacteria than feces of CON-fed infants and did not differ from feces of BF. BF had a higher proportion of bifidobacteria than CON (p=0.0219) and a lower number of Clostridium difficile than FF (p=0.0087). Feces from FF infants had higher concentrations of acetate (p<0.001), butyrate (p<0.001), propionate (p<0.001) and total SCFAs (p=0.0230) than BF; however, fecal pH was lower (p=0.0161) in PRE and BF than CON. Prebiotic supplementation did not alter stool patterns, tolerance, or growth. To conclude, infant formula containing the studied oligosaccharides is well-tolerated, increased abundance and proportion of bifidobacteria, and reduced fecal pH in healthy infants.
Human milk oligosaccharides (HMOs) are abundant in human milk (> 15 g/L) and associated with enhanced intestinal maturation in vitro (Kuntz et al., 2008; Kuntz et al., 2009). Therefore, we aimed to: 1) determine the time course of impact of a model HMO, and; 2) assess the induction of ‘epithelial differentiation’ by three individual HMOs using an in vitro epithelial model of the crypt-villus axis. In study 1, pre-confluent HT-29, pre-confluent Caco-2Bbe, confluent Caco-2Bbe, and post-confluent Caco-2Bbe cells were cultured individually and randomized to 1 of 5 treatments: lacto-N-neotatraose (LNnT) at 0, 100, 200 or 400 mg/L or energy control (0 mg/L plus lactose + N-acetylglucosamine) for 24, 48 or 72 hours of incubation to assess effects on proliferation and differentiation. In study 2, pre-confluent HT-29, pre-confluent Caco-2Bbe, and post-confluent Caco-2Bbe cells were randomized to treatments with one of three HMOs at the following levels for 72 hours of incubation: a) LNnT at 0, 20, 200 or 2000 mg/L or energy control (0 mg /L plus lactose + N-acetyllactosamine); b) 2'-fucosyllactose (2'FL) at 0, 20, 200 or 2000 mg/L or energy control (0 mg/L plus lactose + fucose); c) 6'sialyllactose (6'SL) at 0, 40, 400, 4000 mg/L or energy control (0 mg/L plus lactose + sialic acid). Dependent variables in study 2 included proliferation, differentiation, cell cycle analysis, apoptosis, disaccharidase activity, and absorption and barrier function via modified Ussing chambers. In study 1, compared to 0 mg/L control, LNnT decreased cell proliferation in pre-confluent HT-29 cells at 48 (p<0.05) and 72 hours (p<0.01) and pre-confluent CaCo-2Bbe at 72 hours (p < 0.01). Alkaline phosphatase (AP) activity increased over time and was highest in post-confluent CaCo-2Bbe cells (p<0.001); however, was not affected by treatment at any time point. In study 2, HT-29 cell proliferation was reduced (p<0.05) with 200 and 2000 mg/L LNnT, 2000 mg/L 2'FL, and 400 and 4000 mg/L 6'SL. CaCo-2Bbe cell proliferation was reduced (p<0.05) with 2000 mg/L LNnT, 2000 mg/L 2'FL and 4000 mg/L 6'SL. Reduced proliferation was associated with cell cycle arrest in the G2/M phase in pre-confluent HT-29 cells treated with 200 (p=0.027) and 2000 mg/L (p=0.0033) of LNnT or 2000 mg/L 2'FL (p=0.021) and pre-confluent Caco-2Bbe cells treated with 4000 mg/L 6'SL. Interestingly, in pre-confluent HT-29 cells, treatment with 4000 mg/L 6'SL resulted in S phase arrest (p<0.001), whereas there was a trend (p=0.058) for cell cycle arrest in the G2/M phase with 400 mg/L 6'SL. Differentiation was increased 31% (p=0.030) in HT-29 cells and sucrase activity increased 54% (p=0.036) in post-confluent CaCo-2Bbe cells with 2000 mg/L 2'FL. Transepithelial resistance increased 21% (p=0.002) in post-confluent Caco-2Bbe cells with 200 mg/L LNnT. In summary, inhibition of proliferation was associated with enhanced epithelial differentiation. Well-differentiated cells had greater digestive and barrier function when treated with 2'FL and LNnT, respectively. Further study is needed to assess whether synergist effects occur following concomitant administration of HMOs.
Thus, we evaluated the effects of concomitant administration of 3'-sialyllactose (3'SL), 2'FL, and 6'SL utilizing the same model of the crypt-villus axis discussed above. Cells were randomized to 1 of 18 treatments for 72 hours of incubation: individual HMO treatments at low and high doses (3'SL at 0.2 and 1.0 g/L, 6'SL at 0.4 and 1.0 g/L, 2'FL at 0.2 and 2.0 g/L), concomitant treatments of the previous HMOs at both low and high doses, and controls (0 g/L, media, 4 g/L total HMO, and lipopolysaccharide (LPS)). Pre-confluent HT-29 cells had reduced proliferation (p<0.05) with individual high dose treatments of 6'SL and 2'FL, concomitant administration of the high doses of the HMOs (p<0.05), and total HMO (p<0.001). Pre-confluent CaCo-2Bbe cells were less sensitive to reduction in proliferation where only high dose concomitant treatments containing 2'FL (p<0.01) significantly reduced proliferation in addition to the total HMO (p<0.001). Interestingly, 2'FL did not enhance differentiation in HT-29 cells as we had previously shown; however, it did increase (p<0.05) differentiation in pre-confluent Caco-2Bbe cells. HMO treatments did not enhance AP activity, a general marker of differentiation, nor did it increase disaccharidase activity in post-confluent Caco-2Bbe cells. Disaccharidase activity was only affected by time (p<0.001) in that lactase activity was higher at 12 days as compared to 21 days and the opposite was found for sucrase activity. Apoptosis and necrosis were both significantly decreased (p<0.001) in post-confluent CaCo-2Bbe cells treated with 4.0 g/L HMO as compared to 0 g/L control. In summary, HMO treatment consistently inhibits proliferation with some associated enhancement of epithelial differentiation. Although total pooled HMO reduced apoptosis and necrosis, it is less clear if this is associated with functional outcomes. Further study is needed to determine if these effects translate in vivo; however, results of our in vitro model of the crypt-villus axis suggest addition of HMOs may promote intestinal maturation.